Muscular dystrophy involving loss of the cytoplasmic protein dystrophin (Duchenne Muscular Dystrophy, or DMD) is a devastating disease of both skeletal and cardiac myopathy, with no cure and relatively few effective palliative therapies. Cardiac mortality is now increasingly common, as better care has improved survival into early adulthood. In DMD, dissolution of the dystrophin-sarcoglycan complex results in maladaptive hyper- sensitivity of muscle to mechanical load, involving stretch-responsive mechanisms that increase intracellular calcium and oxidative stress. We recently revealed that transient potential receptor cation-6 (TRPC6) ion channel is a major mechano-sensor in DMD myocardium (mdx/utrophin+/- mice), regulating amplified force, Ca2+ and arrhythmia in contracting DMD cardiac myocytes subjected to auxotonic stress. Acute activation of protein kinase G1a (PKG1?) potently blocks this response in a TRPC6-dependent manner, and in another new study, we showed that if PKG1a becomes oxidized, it is less effective in countering TRPC6 signaling. As DMD invokes oxidant stress and reduces nitric oxide (NO) signaling in muscle, the change in PKG1? redox could contribute to worsened disease. In a third study reported in Nature, we revealed a new therapeutic option by showing that PDE9A targets natriuretic peptide not NO derived cGMP - and its inhibition circumvents blunted NO-signaling and oxidant stress and improves cardiac responses to stress. As natriuretic peptide levels are elevated in DMD patients and experimental models, this signaling cascade may be very relevant. The current project synthesizes these exciting new discoveries, and using our sophisticated cell-based mechano-sensing and imaging and in vivo models, will address several novel hypotheses. First, we test that TRPC6 interacts with other critical determinants of calcium mishandling in DMD, including Ca2+ leak from the sarcoplasmic reticulum and sodium/calcium exchange, as key contributors to its adverse impact. We then test if PKG activation reverses these changes. Second, we determine if TRPC6 activation drives or is in driven by oxidant stress particularly generated by NADPH-oxidase 2 (NOX2), with which it can co-localize. Here we employ novel genetically encoded sensors of redox localized to NOX2, caveolae, and cytosol. We also test if PKG1? oxidation contributes to TRPC6 activation in DMD, employing knock-in mouse models with a redox-dead PKG1? mutation (C42S). Lastly, we perform pre-clinical studies to test if either chronic gene deletion of Trpc6 or pharmacological PDE9A inhibition ameliorates DMD cardiac disease, improving myocyte function and reversing abnormal mechano-stimulation and arrhythmia. Together, these studies will forge major new insight into DMD cardiac disease, focusing on TRPC6 and its coupling to calcium and ROS dysregulation, and establish if a new therapeutic approach involving PDE9A inhibition will benefit this disease.